Method for Preparing Cell Fraction Containing Hemangioblasts

ABSTRACT

Mouse PCLP1 was identified by expression cloning with the use of a monoclonal antibody against a surface antigen of a cell line derived from mouse AGM. By fractionating PCLP1-positive/CD45-negative cells and culturing them in vitro, it was clarified that these cells differentiate into endothelial-like cells, angioblast-like cells, and hematopoietic cells. By transferring the PCLP1-positive/CD45-negative cells into a mouse defective in the hematopoietic function, the hematopoietic system was reconstructed over a long period of time. These facts indicate that the PCLP1-positive/CD45-negative cells contain mammalian hemangioblasts capable of expressing the activity as long-term repopulating hematopoietic stem cells (LTR-HSC). The present invention provides a method for preparing a cell fraction containing hemangioblasts, the cell fraction prepared by the method, and use of this cell fraction.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/130,076, filed May 10, 2002, which claims benefit of priority toJapan Patent Application No. 11-320234, filed Nov. 10, 1999, and PCTInternational Application No. PCT/JP00/07817, filed Nov. 7, 2000.

TECHNICAL FIELD

The present invention relates to a marker molecule for hemangioblasts,method for preparing a cell fraction containing hemangioblasts using themarker molecule, cell fraction prepared by the method, and use of thecell fraction.

BACKGROUND ART

Development of hematopoiesis proceeds through two distinct steps, i.e.primitive and definitive hematopoiesis. In mice, primitive hematopoiesisbegins in the extraembryonic yolk sac at 7.5 days post coitum (dpc) ingestation, while definitive hematopoiesis, which is distinguished byenucleated erythrocytes, lymphopoiesis, and generation of long termrepopulating hematopoietic stem cells (LTR-HSCs), originates from theintraembryonic aorta-gonad-mesonephros (AGM) region at 10.5 to 11.5 dpc(Muller, A. M. et al. (1994) Immunity, 1, 291-301) (also reviewed by(Dzierzak, E. et al. (1998) Immunol. Today 19, 228-236; Keller, G. etal. (1999) Exp. Hematol. 27, 777-787). Within 1 to 3 days of theiremergence, LTR-HSCs migrate from the AGM region to the fetal liver andthen emigrate to the bone marrow just before birth. Lymphopoietic cellsand multi-potential hematopoietic progenitors are also detected in thepara-aortic splanchnopleura (P-Sp) region of mouse embryos at 7.5 to 9.5dpc (Cumano, A. et al. (1996) Cell 86, 907-916; Delassus, S., andCumano, A. (1996) Immunity 4, 97-106; Godin, I. et al. (1995) Proc.Natl. Acad. Sci. USA 92, 773-777), an intraembryonic site preceding theAGM region. However, LTR-HSCs, which are capable of repopulatinglethally irradiated adult mice, have not been found in the P-Sp region.Interestingly, it was recently reported that LTR-HSCs can be detected inthe yolk sac and the P-Sp region after transplantation into the liversof busulfan-treated newborn mice (Yoder, M. C. et al. (1997) Immunity 7,335-344). Therefore, one speculation has been that LTR-HSCs generated inthese sites lack homing capacity to the bone marrow and that thephenotypic differences in hematopoiesis between the yolk sac, the P-Spregion, and the AGM region can be mostly attributed to the supportingmicroenvironment. However, it still remains unknown how LTR-HSCs in theyolk sac acquire full repopulation activity.

Early in the last century, detailed observations of the earlydevelopment of chick embryos led to the hypothesis that hematopoieticcells and endothelial cells arise from a common precursor termed thehemangioblast (Murray, P. D. F. (1932) Proc. Roy. Soc. London 11,497-521; Sabin, F. R. (1920) Contributions to Embryology 9, 213-262)[also reviewed by (Wagner, R. C. (1980) Adv. Microcirc. 9, 45-75)]. Inthe last 5 years, a number of studies have provided evidence supportingthis hypothesis. First, a series of elegant grafting experiments usingchicks and quails demonstrated that the splanchnopleural mesoderm isable to generate hematopoietic cells and endothelium, while the paraxialmesoderm lacks this hematogenic capacity (Pardanaud, L. et al. (1996)Development 122, 1363-1371). Hematogenic activity in the former regionis regulated by endoderm-derived cytokines such as vascular endothelialgrowth factor (VEGF), basic fibroblast growth factor (bFGF), andtransforming growth factor β1 (TGFβ1), whereas ectodermal factors suchas epidermal growth factor (EGF) suppress it in the latter region(Pardanaud, L. and Dieterlen-Lievre, F. (1999) Development 126,617-627). In the splanchnopleural mesoderm, cells expressing VEGFreceptor 2 (VEGF-R2) were shown to form both hematopoietic andendothelial colonies (Eichmann, A. et al. (1997) Proc. Natl. Acad. Sci.USA 94, 5141-5146). Furthermore, endothelial cells in the dorsal aortagenerated CD45⁺ hematopoietic cells in vivo as evidenced by celllabeling experiments using DiI-labeled acetylated low densitylipoprotein (DiI-Ac-LDL) (Jaffredo, T. et al. (1998) Development 125,4575-4583).

While similar in vivo grafting experiments are not possible in mammaliansystems, it was found that hematopoietic cells were clustered at theventral wall of the dorsal aorta in a 5 week-old human embryo (Tavian,M. et al. (1996) Blood 87, 67-72). More recently, Tavian et al. showedthat CD34⁺ cells in the dorsal aorta and vitelline artery of humanembryos are capable of generating hematopoietic cells in vitro (Tavian,M. et al. (1999) Development 126, 793-803). In mice, Nishikawa et al.recently showed that cells expressing Flk1 (mouse counterpart ofVEGF-R2) and vascular endothelial cadherin (VECadherin) in the yolk sacand the P-Sp region of mouse embryos at 9.5 dpc gave rise tolymphohematopoietic cells in vitro (Nishikawa, S. et al. (1998) Immunity8, 761-769). Similarly, Flk1⁺ hematogenic endothelial cells weregenerated from ES cells in vitro (Choi, K. et al. (1998) Development125, 725-732; Nishikawa, S. I. et al. (1998) Development 125,1747-1757). The idea that putative hemangioblasts express Flk1 aroseoriginally from the finding that knockout mice lacking Flk1 exhibitedsevere defects in both hematopoiesis and vasculogenesis in the yolk sac(Shalaby, F. et al. (1995) Nature 376, 62-66). Furthermore, Flk1-nullcells did not contribute to definitive hematopoiesis (Shalaby, F. et al.(1997) Cell 89, 981-990), although a recent report suggested that asignificant number of hematopoietic cells can be induced from Flk1-nullES cells in vitro (Schuh, A. C. et al. (1999) Proc. Natl. Acad. Sci. USA96, 2159-2164).

In conformity with the critical role of TGFβ1 in the induction ofhematopoiesis and vasculogenesis in chick embryos, knockout micedeficient in TGFβ1 also exhibited severe defects in both systems(Dickson, M. C. et al. (1995) Development 121, 1845-1854). Furthermore,gene disruption of the SCL/Tal-1 transcription factor caused defects inboth hematopoiesis and vasculogenesis, which were similar to those ofFlk1 knockout mice (Porcher, C. et al. (1996) Cell 86, 47-57: Visvader,J. E. et al. (1998) Genes Dev. 12, 473-479). Mutant zebrafish devoid ofthe SCL/Tal-1 gene also showed similar defects (Liao, E. C. et al.(1998) Genes Dev. 12, 621-626) and forced expression of SCL/Tal-1 inzebrafish embryos resulted in the overproduction of hematopoietic andvascular cells (Gering, M. et al. (1998) EMBO J. 17, 4029-4045). Takentogether, these studies clearly demonstrate the presence ofhemangioblasts, the common precursors of both hematopoietic andendothelial cells, in fish, avian, and mammalian embryos and thatVEGF-R2/Flk1, TGFβ1, and SCL/Tal-1 are essential for the development ofhemangioblasts. However, the nature of hemangioblasts remainsunexplored, in particular, there is yet no absolute evidence thatLTR-HSCs are derived from hemangioblasts.

SUMMARY OF THE INVENTION

An objective of the present invention is to provide a novel markermolecule for hemangioblasts, method for preparing a cell fractioncontaining hemangioblasts using the marker molecule, cell fractionprepared by the method, and use of the cell fraction.

The Inventors characterized the nature of mammalian hemangioblasts byusing their AGM primary culture system, in which multipotentialhematopoietic progenitor cells and endothelial-like cells expand invitro (Mukouyama, Y et al. (1998) Immunity 8, 105-114). In this culture,oncostatin M (OSM), a member of the IL-6 family of cytokines, isessential for the expansion of both cell types. Although OSM plays anessential role in the expansion of both cell populations, OSM does notdirectly stimulate the growth of hematopoietic progenitors in colonyforming assays (data not shown). The inventors thus hypothesized thatendothelial-like cells may contain hemangioblasts that producehematopoietic progenitors in the AGM culture.

In order to isolate a novel marker molecule of endothelial-like cells inAGM culture, the present inventors prepared a monoclonal antibodyagainst endothelial-like cells and conducted expression cloning usingthe antibody. As a result, the inventors succeeded in cloning a geneencoding the mouse counterpart (mouse PCLP1) corresponding to human andrabbit podocalyxin-like protein 1 (PCLP1).

Furthermore, the present inventors investigated properties of PCLP1⁺CD45cells in the AGM region to find out the formation of both hematopoieticcells and endothelial cells from PCLP1⁺CD45⁻ cells in vitro. Byinjecting PCLP1⁺CD45⁻ cells into neonatal livers ofbusulfan-administered mice, the inventors further demonstrated that aplurality of hematopoietic cell lineages are produced over a long periodof time in vivo, thereby proving for the first time that mammalianhemangioblasts are capable of constructing LTR-HSCs.

More specifically, PCLP1⁺CD45⁻ cells thus selected exhibitedendothelial-like morphology, incorporated acetylated low-densitylipoprotein, and proliferated in response to OSM. In the co-presence ofOP9 stromal cells together with VEGF and OSM, almost all the PCLP1⁺CD45⁻cells became positive for CD34, CD31 and VECadherin (FIG. 15), acquiringa capability to form a cellular network in the matrigel substrate (FIG.16). These results indicate that PCLP1⁺CD45⁻ cells have angioblastactivity, and that OSM is essential for their proliferation anddifferentiation. On the other hand, Dil⁺CD45⁻ endothelial-like cells(cf. examples) in the AGM primary culture generated hematopoietic cellsin vitro (FIG. 1), and PCLP1⁺CD45⁻ cells selected above similarlygenerated hematopoietic cells in vitro in the presence of hematopoieticgrowth factors and OP9 cells (FIG. 17). Most important finding was thatPCLP1⁺CD45⁻ cells reconstructed the entire hematopoietic system over along period of time when transplanted into busulfan-treated neonatalmice (FIGS. 18 to 21, and Table 1).

Thus, the present invention identifies PCLP1 as a novel cell marker fordiscriminating a cell fraction containing hemangioblasts and provides amethod for preparing a cell fraction containing hemangioblasts usingthis marker.

As described above, although Nishikawa et al. previously reported thepossibility that Flk1 and VECadherin could be marker molecules forhemangioblasts (Nishikawa, S. I. et al. (1998) Development 125,1747-1757; Nishikawa, S. I. et al. (1998) Immunity 8, 761-769),expression level of PCLP1 is extremely high compared to those of Flk1and VECadherin, which makes PCLP1 an excellent marker for firstseparating hemangioblasts.

More specifically, the present invention comprises:

(1) a method for preparing a cell fraction containing hemangioblasts,wherein said method comprises separating cells comprising aPCLP1-positive phenotype,

(2) the method according to (1), which further comprises separatingcells comprising a CD45-negative phenotype,

(3) the method according to (1) or (2), wherein said cells are separatedfrom cells derived from the aorta-gonad-mesonephros (AGM) region,

(4) a PCLP1-positive cell fraction containing hemangioblasts that isprepared by a method according to any one of (1) through (3),

(5) the cell fraction according to (4), which generates or containslong-term repopulating hematopoietic stem cells (LTR-HSCs).

(6) a cell composition containing the cell fraction according to (4) anda culture medium,

(7) a method for preparing a chimeric animal, wherein said methodcomprises transplanting the cell fraction according to (4),

(8) a chimeric animal transplanted with the cell fraction according to(4),

(9) the chimeric animal according to (8), wherein donor (transplantedcells)-derived blood cells can be reconstructed,

(10) the chimeric animal according to (8) or (9), wherein said animal ismouse,

(11) a DNA according to any one of the following (a) through (c),wherein the DNA encodes the mouse-derived PCLP1 protein:

-   -   (a) a DNA comprising the coding region of the nucleotide        sequence set forth in SEQ ID NO: 1,    -   (b) a DNA encoding a protein comprising the amino acid sequence        set forth in SEQ ID NO: 2, and    -   (c) a DNA encoding a protein comprising the amino acid sequence        set forth in SEQ ID NO: 2 in which one or more amino acids are        substituted, deleted, inserted, and/or added,

(12) a protein encoded by the DNA according to (11),

(13) a vector into which the DNA according to (11) has been inserted,

(14) a host cell carrying the vector according to (13),

(15) a method for preparing the protein according to (12), wherein saidmethod comprises the steps of culturing the host cell according to (14)and collecting expressed proteins from said host cell or culturesupernatant thereof,

(16) an antibody against PCLP1 protein, wherein the antibody is used fordetecting or separating a cell fraction containing hemangioblasts,

(17) the antibody according to (16) that binds to a protein as definedin (12),

(18) a separation reagent for a cell fraction containing hemangioblasts,wherein said reagent comprises the antibody according to (16) or (17),

(19) an antibody that specifically binds to mouse-derived PCLP1 protein,

(20) an antibody that binds to a peptide comprising the amino acidresidues at positions 1 to 405 in the amino acid sequence set forth inSEQ ID NO: 2,

(21) a peptide containing a partial sequence comprising at least 7 ormore consecutive amino acid residues at positions 1 to 405 in the aminoacid sequence set forth in SEQ ID NO: 2, and,

(22) a polynucleotide comprising at least 15 nucleotides, wherein thepolynucleotide is complementary to DNA comprising the nucleotidesequence set forth in SEQ ID NO: 1 or to the complementary strandthereof, and is used in the amplification and detection of theexpression of the DNA according to (11), or in the expression control ofthe DNA.

Besides mouse PCLP1, “PCLP1s” of the present invention includes, unlessthe origin is otherwise specified, PCLP1s derived from vertebratesincluding human or rabbit podocalyxin-like protein 1 (PCLP1) (Kershaw,D. B. et al. (1997) J. Biol. Chem. 272, 15708-15714; Kershaw, D. B. etal. (1995) J. Biol. Chem. 270, 29439-29446), preferably PCLP1s derivedfrom mammals. Similarly, “CD45s” of the present invention includes CD45sderived from vertebrates, preferably CD45s derived from mammals.

Herein, “hemangioblasts” refers to cells capable of generating bothendothelial cells and hematopoietic cells.

“Endothelial cells” refers to adherent cells showing endothelial cellmorphology, namely polygonal morphology when cultured in vitro, whichhave the activity to incorporate acetylated low density lipoprotein. Inthis invention, more preferably, endothelial cells can proliferate inresponse to stimulation by OSM. Even more preferably, in endothelialcell differentiation culture systems, cells positive for hematopoieticcell markers such as CD34, CD31, and VECadherin could be generated whenco-cultured with OP9 stromal cells in the presence of VEGF, OSM, etc.Also, still more preferably, endothelial cells can give rise to acellular network formation in matrigel plate assays. These propertiescan be assayed according to methods described in Examples.

“Hematopoietic cells” refers to cells expressing a hematopoietic cellphenotype, meaning spherical non-adherent cells with a CD45- orTer119-positive phenotype. During their differentiation, hematopoieticcells specifically express combinations of B220, Mac-1, Gr-1, Thy-1,CD4, CD8, etc. as lineage markers.

“Hematopoietic cells” includes “hematopoietic stem cells”. Thesehematopoietic stem cells are preferably CD45⁻ positive. Preferably, whenco-cultured with OP9 cells in the presence of hematopoietic growthfactors including SCF, interleukin (IL)-3, erythropoietin (EPO), etc.,hematopoietic stem cells generate myeloid (e.g. Mac-1/Gr-1-positive),lymphoid (e.g. B220/Thy-1-positive) or erythroid (e.g. Ter119-positive)cells. Alternatively, hematopoietic stem cell phenotype can be confirmedby reconstruction of hematopoietic stem cells or blood cells derivedfrom transplanted cells that are transplanted into animals lackinghematopoietic functions.

In this invention, “long term repopulating hematopoietic stem cells(LTR-HSCs)” refers to hematopoietic stem cells capable of reconstructinghematopoiesis over a long duration.

The present invention provides a method for preparing a cell fractioncontaining hemangioblasts, characterized by separating cells expressinga PCLP1 (podocalyxin-like protein 1)-positive (described as PCLP⁺)phenotype.

As cells used in the aforementioned separation, tissues and cellspresumed to contain hemangioblasts, or hemangioblast cultures, may beused. Most preferable cells are those derived from theaorta-gonad-mesonephros (AGM) region. These cells may bevertebrate-derived, preferably mammalian cells (for example, cells fromrodents), or may be human cells. In mice, cells derived from theintraembryonic AGM region at 10 to 11.5 dpc, which corresponds to theAGM region of a 4 to 5 week-old human embryo, are preferable inparticular.

Moreover, since it has been suggested that dorsal aorta-associatedendothelial cells contain the budding site of hematopoietic cells(Tavian, M. et al. (1996) Blood 87, 67-72; Tavian, M. et al. (1999)Development 126, 793-803), hemangioblasts are highly likely to exist inthis region. Furthermore, the present inventors indicate the possibilityof the presence of hemangioblasts in the genital ridge region (F, I, Oin FIG. 10 to 11). Therefore, cells derived from these regions can bealso used.

OSM-dependant proliferation of hematopoietic precursor cells andendothelial-like cells were also observed in a primary culture of theP-Sp region of an embryo at 9.5 dpc similarly to a culture of AGMderived from a mouse embryo at 11.5 dpc. Although such a proliferationwas not observed in the yolk sac at 9.5 dpc, PCLP1⁺CD45⁻ cells werepresent in both yolk sac and P-Sp region (data not shown). Therefore,cells derived from the yolk sac and P-Sp region are highly likely tocontain hemangioblasts. Therefore, cells such as these can also be usedin the separation in this invention.

It is also possible to isolate PCLP1⁺ cells or PCLP1⁺CD45⁻ cells usingES-derived cells. Generation and proliferation of LTR-HSC from ES cellsare also important in the application of human ES cells. Furthermore,recently, CD34⁺ blood progenitor endothelial cells were found inperipheral blood, which were shown to be bone marrow-derived (Asahara,T. et al. (1997) Science 275, 964-967; Takahashi, T. et al. (1999) Nat.Med. 5, 434-438). Very recently, Bjornson et al. reported that LTR-HSCsare derived from cultured neural stem cells in vivo (Bjornson, C. R. etal. (1999) Science 283, 534-537). Therefore, it is likely thathemangioblasts exist in these tissues besides embryonic hematopoieticsites. In this invention, such cells may also be also used for isolatingPCLP1⁺ cells or PCLP1⁺CD45⁻ cells.

Neonatal and adult tissues, for example, umbilical cord and bone marrowcan be also used. Separation of hemangioblasts using the method of thisinvention is extremely significant in the clinical application of thesecells.

Separation of PCLP1⁺ cells can be performed, for example, by cellsorting as described in Examples using anti-PCLP1 antibody.

In this invention, in order to obtain cell fractions containinghemangioblasts in a high concentration, it is preferable to separatecells further expressing the CD45⁻ phenotype in addition to the PLCP1⁺phenotype. Separation of cells having the PLCP1⁺CD45⁻ phenotype can beperformed, for example, according to methods described in Examples.PLCP1⁺ cell fraction or PCLP1⁺CD45⁻ cell fraction containinghemangioblasts can be further subdivided using different cell markers.

For example, in the present invention, it may be useful to fractionateCD34⁺ cells from PCLP1⁺ cell fraction or PCLP1⁺CD45⁻ cell fraction. InExamples, the inventors have identified PCLP1 as a marker forhemangioblasts. PCLP1 is a highly glycosylated protein with somesimilarity to CD34, a conventional marker for LTR-HSCs. Interestingly,both PCLP1 and CD34 are ligands for L-selectin in the lymphocyte-highendothelium venule and contain conserved amino acid sequences in theircytoplasmic regions (Sassetti, C. et al. (1998) J. Exp. Med. 187,1965-1975), suggesting an overlapping function between the twomolecules. In fact, both PCLP1 and CD34 are expressed in the dorsalaortic endothelium and the genital ridge region (FIGS. 10 and 11) and asmuch as 91% of CD34⁺ cells in the AGM region also express PCLP1 (FIG. 8,right). In chicken, thrombomucin, an avian counterpart of PCLP1, isexpressed in hematopoietic progenitors and thrombocytes (McNagny, K. M.et al. (1997) J. Cell Biol. 138, 1395-1407), however no avian CD34 hasbeen identified. Therefore, the functions, if any, of CD34 might becompensated by PCLP1. Sanchez et al. showed that LTR-HSCs in the AGMregion are CD34⁺c-Kit⁺ (Sanchez, M. J. et al. (1996) Immunity 5,513-525) and LTR-HSCs in the yolk sac and P-Sp region were also found inthe CD34⁺c-Kit⁺ fraction (Yoder, M. C. et al. (1997) Immunity 7,335-344). Since PCLP1⁺CD34⁺CD45⁻ cells (12% of the PCLP1⁺CD45⁻ cells)exist in the AGM region (FIG. 8, right), the LTR-HSC activity found inthe CD34 fraction might represent, in part, that of the hemangioblasts.Thus, the PCLP1⁺CD34⁺CD45⁻ cells in the AGM region are important as acellular fraction containing hemangioblasts, and are likely to contain ahigh concentration of LTR-HSCs that are capable of reconstructing, inparticular, hematopoietic systems.

Furthermore, in this invention, it may be also useful to fractionateFlk1-positive cells from the PCLP1⁺ cell fraction or the PCLP1⁺CD45⁻cell fraction. Recently, Nishikawa et al. demonstrated the hematopoieticactivity in an Flk1⁺VECadherin⁺CD45⁻ cell population derived from theyolk sac and P-Sp region of mouse embryo at 9.5 dpc (Nishikawa, S. I. etal. (1990) Immunity 8, 761-769). Studies on ES differentiation in vitroand Flk1-knockout mice also indicated that putative hemangioblastsexpress Flk1 (Choi, K. et al. (1998) Development 125, 725-732;Nishikawa, S. I. et al. (1998) Development 125, 1747-1757; Shalaby, F.et al. (1995) Nature 376, 62-66). In accordance with these results, onefraction (12%) of PLCP1⁺CD45⁻ cells in the AGM region expressed Flk1(FIG. 9, left), and PLCP1⁺CD45⁻ cells cultured in the presence of OSMwere all Flk1-positive (FIG. 14, below). In contrast, frequency ofVECadherin⁺ cells in the PLCP1⁺CD45⁻ cell population in the AGM regionwas very low (3%) (FIG. 9, right). From these findings, PLCP1⁺CD45⁻Flk1⁺cells in AGM region are important as a cell fraction containinghemangioblasts.

Cell fractions prepared in the present invention can be cultured orstored in an appropriate medium, which may be supplemented with serum,growth/differentiation factors, etc. As a medium, for example, DMEMcontaining 15% FCS supplemented with OSM and SCF, or the like can beused.

Among cells contained in cell fractions of this invention, long-termrepopulating hematopoietic stem cells (LTR-HSCs) are important inparticular. Presence of LTR-HSC in a cell fraction can be confirmed bytransplanting the cell fraction into an animal made deficient inhematopoietic functions to prepare a chimeric animal, and assaying thecapability of the cells to reconstruct the hematopoietic system.

As described in Examples, chimeric animals can be prepared bytransplanting the cell fraction of this invention into livers ofneonatal mice in which the hematopoietic function has been destructed bybusulfan administration.

There is no restriction on the type of animals used for preparingchimeras, examples being mice, rabbits, other large-sized animals, etc.

Establishment of chimerism can be confirmed by examining thepost-transplantational generation of donor-derived blood cells.

In chimeric animals transplanted with the cell fraction of thisinvention containing LTR-HSCs, donor (transplanted cells)-derivedLTR-HSC is generated in the recipient reconstructing the hematopoieticsystem. Inclusion of LTR-HSC in transplanted cells can be confirmed bythe appearance of donor-derived lymphoid, myeloid, and erythroid cellsabove the detection limit (for example, 1% or more) in recipients. Inaddition, occurrence of a long-term reconstruction of hematopoiesis canbe confirmed by detecting donor-derived blood cells at least 60 days,more preferably 180 days after the transplantation of LTR-HSC.

PCLP1⁺CD45⁻ cells and recipients transferred with the cells are usefulin the screening of drugs that control the proliferation anddifferentiation of hemangioblasts. For example, by adding a testcompound to PCLP1⁺CD45⁻ cells in culture, effects of the compound on theproliferation, differentiation, hematopoietic function, and the like ofthe cells can be examined. By administering a test compound to arecipient (for example, mouse) transplanted with PCLP1⁺CD45⁻ cells,effects of the compound on hematopoiesis of the recipient can be alsoinvestigated.

Furthermore, the present invention relates to an antibody against thePCLP1 protein used for the detection or separation of cell fractionscontaining hemangioblasts. This invention also relates to the use of anantibody raised against the PCLP1 protein in the detection or separationof cell fractions containing hemangioblasts. Such an antibody binds tothe cell surface PCLP1 protein. Such an antibody is suitably prepared byusing the extracellular domain of the PCLP1 protein as an antigen, orcells expressing the PCLP1 protein as an immunogen as described inExamples.

There is no particular restriction on the animal species from which thePCLP1 protein that is used as the immunogen is derived, and it maybehuman (J. Biol. Chem. 272: 15708-15714 (1997)), mouse, rat (Accessionnumber: ABO20726), rabbit (J. Biol. Chem. 270: 29439-29446 (1995)),chicken (J. Cell Biol. 138: 1395-1407 (1997)), or another vertebrate.Antibodies can be prepared according to methods well-known in the field.For example, in the case of a monoclonal antibody, it may be prepared bythe rat footpad immunization method (Hockfield, S. et al. (1993)“Selected Methods for Antibody and Nucleic Acid Probes”, Volume 1 (NewYork: Cold Spring Harbor Laboratory Press)), etc.

Such an antibody can be appropriately combined with buffers andstabilizers to make a reagent for detecting or separating cell fractionscontaining hemangioblasts. This antibody may also become a test reagentfor cell fractions containing hemangioblasts. The antibody may befluorescence-labeled.

The present invention also provides the mouse PCLP1 protein. The mousePCLP1 gene was isolated by expression cloning using a monoclonalantibody against the cell surface antigen on a mouse AGM region-derivedcell line (LO cells). The nucleotide sequence of cDNA encoding the mousePCLP1 protein isolated by the present inventors is set forth in SEQ IDNO: 1, and the amino acid sequence of the mouse PCLP1 protein encoded bythe cDNA is set forth in SEQ ID NO: 2, respectively. PCLP1⁺ cellfractions contain hemangioblasts capable of generating endothelial cellsand hematopoietic cells.

Proteins of this invention include, as long as they can serve as markermolecules for hemangioblasts (for example, proteins with overlappingantigenicities), not only the wild type PCLP1 protein (SEQ ID NO: 2),but also proteins structurally analogous thereto. Such structurallyanalogous proteins include mutants of the wild type mouse PCLP1 protein.Whether antigenicity is overlapped or not can be determined byimmunizing a recipient animal with the wild type mouse PCLP1 protein asantigen and examining whether the antibody thus produced has reactivitytoward a protein of interest. Alternatively, the above test can beperformed by preparing an antibody against a protein of interest todetermine the reactivity of the protein with the wild type mouse PCLP1protein.

Such proteins include not only naturally occurring mutants but alsoartificially prepared mutants that can be produced by those skilled inthe art, for example, using the known method for mutagenesis. Methodsknown to those skilled in the art for modifying amino acids in proteinsare exemplified by Kunkel's method and PCR.

In artificial alteration of amino acids in proteins, the number of aminoacid residues to be altered is usually 30 or less, preferably 10 orless, and more preferably 5 or less. An amino acid having propertiessimilar to those of the amino acid to be substituted is preferably usedfor substitution. For example, since Ala, Val, Leu, Ile, Pro, Met, Phe,and Trp are all classified into the non-polar amino acid group, they areconsidered to have similar properties. Non-charged amino acids includeGly, Ser, Thr, Cys, Tyr, Asn, and Gln. Acidic amino acids include Aspand Glu, while basic amino acids include Lys, Arg, and His.

The protein of this invention can be prepared as either a naturalprotein or a recombinant protein utilizing gene recombinationtechniques. Natural proteins can be prepared by, for example, subjectingextracts from tissues and cells that are presumed to express mouse PCLP1protein (for example, LO cells, embryo cells in AGM region, etc.) toaffinity chromatography using the above-described antibody to the mousePCLP1 protein. On the other hand, recombinant proteins may be preparedby culturing cells transformed with DNA encoding the mouse PCLP1protein, allowing the transformants to express the protein, andrecovering the protein as described below. Proteins of this inventioncan be fused to peptide tags and other proteins. Such fusion proteinscan be useful for facilitating the purification and detection ofproteins.

The present invention also includes partial peptides of the protein ofthis invention. Partial peptides of this invention include peptidescontaining partial sequences comprising at least 7 or more consecutiveamino acid residues, preferably 8 amino acid residues or more, and morepreferably 9 amino acid residues or more in the region specific (atpositions 1 to 405 of the amino acid sequence set forth in SEQ ID NO: 2)to mouse PCLP1. An antibody specifically binding to the mouse-derivedPCLP1 protein can be obtained by preparing the antibody using theabove-described partial peptides as antigens. Examples of partialpeptides of this invention are, for example, those of the N-terminalregion of proteins of this invention (for example, SEQ ID NO: 2) andintracellular domain thereof, and these can be used in the antibodypreparation. Furthermore, in the mouse PCLP1 protein (SEQ ID NO: 2),peptides containing the regions comprising 12 amino acid residues atpositions 440 to 451, 10 amino acid residues at positions 464 to 473, or4 amino acid residues at positions 500 to 503 in the intracellulardomain analogous to CD34 are considered to be useful as partial peptidesto search functions common to both CD34 and PCLP1. Partial peptides ofthis invention can be produced by, for example, genetic engineeringtechniques, known peptide synthesis methods, or by digestion of theprotein of this invention with appropriate peptidases.

This invention also relates to DNAs encoding the proteins of theinvention. DNAs encoding the protein of this invention are notparticularly limited as long as they can encode the proteins of thisinvention, including cDNAs, genomic DNAs, and synthetic DNAs. DNAshaving any desired nucleotide sequence based on the degeneracy ofgenetic code are also included in this invention as long as they canencode the proteins of this invention.

cDNAs encoding the proteins of this invention can be screened, forexample, by labeling cDNA of SEQ ID NO: 1 or segments thereof, RNAscomplementary to them, or synthetic oligonucleotides comprising partialsequences of the cDNA with ³²P, etc., and hybridizing them with a cDNAlibrary derived from tissues (e.g., cells derived from AGM region ofembryo, etc.) expressing the proteins of this invention. Alternatively,such cDNAs can be cloned by synthesizing oligonucleotides correspondingto nucleotide sequences of these cDNAs, and amplifying them by PCR withcDNA derived from suitable tissues (e.g. cells derived from AGM regionof embryo, etc.) as a template. Genomic DNA can be screened, forexample, by labeling cDNA of SEQ ID NO: 1 or segments thereof, RNAscomplementary to them, or synthetic oligonucleotides comprising partialsequences of the cDNA with ³²P, etc., and hybridizing them with agenomic DNA library. Alternatively, the genomic DNA can be cloned bysynthesizing oligonucleotides corresponding to nucleotide sequences ofthese cDNAs, and amplifying them by PCR with genomic DNA as a template.Synthetic DNAs can be prepared, for example, by chemically synthesizingoligonucleotides comprising partial sequences of cDNA of SEQ ID NO: 1,annealing them to form a double strand, and ligating them with DNAligase.

These DNAs are useful for the production of recombinant proteins. Aprotein of this invention can be prepared as a recombinant protein byinserting the DNA encoding the protein (e.g. DNA of SEQ ID NO: 1) intoan appropriate expression vector, transforming suitable cells with thevector, culturing the transformants, and purifying the expressed proteinfrom the transformants or their culture supernatant.

There is no limitation on the host and vector to be used, and it ispossible to use a prokaryotic or eukaryotic system. Specifically, forexample, COS7 cells and the pME18S vector can be used. A vector can beintroduced into cells by a known method, for example, DEAE-dextranmethod (Blood 90: 165-173, 1997) for mammalian cells.

Recombinant proteins expressed in host cells can be purified by knownmethods. The protein of this invention expressed in the form of a fusionprotein, for example, with a histidine residue tag orglutathione-S-transferase (GST) attached to the N-terminus can bepurified by a nickel column or a glutathione sepharose column, etc.

The present invention also provides a polynucleotide containing at least15 nucleotides complementary to the DNA comprising the nucleotidesequence of SEQ ID NO: 1 or to the complementary strand thereof, whichis used to amplify a DNA encoding a mouse PCLP1 protein, detect theexpression, or regulate the expression. Herein, the term “complementarystrand” refers to one strand of a double strand polynucleotidecomprising A:T (A:U) and G:C base pairs, when viewed against the otherstrand. Furthermore, “complementary” means not only when a nucleotidesequence is completely complementary to a continuous nucleotide sequencewith at least 15 nucleotides, but also when there is an identity of atleast 70%, preferably at least 80%, more preferably 90%, and much morepreferably 95% or more at the nucleotide sequence level. The identitycan be determined by BLAST. Polynucleotides include DNA and RNA.Nucleotide derivatives can also be included.

Such polynucleotides include probes, primers, nucleotides or nucleotidederivatives (e.g. antisense oligonucleotides and ribozymes, etc.), whichcan specifically hybridize with DNAs encoding mouse PCLP1 proteins, orDNAs complementary to said DNAs.

cDNAs encoding the proteins of this invention or oligonucleotidescomprising partial sequences thereof can be used for cloning genes andcDNAs encoding the proteins of this invention, or amplifying them byPCR. They can also be used as, for example, probes for Northern blotanalyses, or as primers for RT-PCR to detect or quantify the expression.The cDNAs and oligonucleotides can also be utilized for detectingpolymorphism or an aberration (gene diagnosis, etc.) of the gene or cDNAby the restriction fragment length polymorphism (RFLP) method, singlestrand DNA conformation polymorphism (SSCP) method, etc. It is alsopossible to suppress the expression of the mouse PCLP1 protein using anantisense polynucleotide.

The present invention also provides an antibody specific to mouse PCLP1.The antibody may be prepared by extracting sequences specific to themouse PCLP1 protein based on a comparison of the amino acid sequence ofmouse PCLP1 protein with those of PCLP1 proteins derived from othervertebrates, and immunizing appropriate animals with peptides havingsaid sequences as described above. There is no limitation in the regionof these peptides that is used for the immunization as long as they areimmunogenic. Antibodies of this invention include, for example, thosebinding to peptides comprising the amino acid residues at positions 1 to405 set forth in SEQ ID NO: 2.

Antibodies thus prepared can be utilized, besides in cell sorting, forexample, in the affinity purification of proteins of this invention,test and diagnosis of disorders in patients or in disease models havingdisorders caused by an expressional aberration, or structuralabnormality of a protein of this invention, and the like, and also inthe detection of the expression levels of the inventive proteins. Morespecifically, aberrations in the expression and structure of proteins ofthis invention can be tested and diagnosed by using methods such asFACS, Western blotting, immunoprecipitation, ELISA, immunohistochemicaltechnique, etc. to detect the proteins in samples extracted fromtissues, blood, cells, or tissue segments. Prior arts and otherreferences cited in the present specification are all incorporatedherein as a part thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram representing generation of hematopoietic cells fromendothelial-like cells in an AGM culture. Endothelial-like cellsgenerated in a day 6 AGM primary culture were pulse labeled withDiI-Ac-LDL for 6 h. DiI⁺CD45⁻ cells (RI fraction) were then isolated byFACS and inoculated into an unlabeled day 6 AGM culture. After a 4-daychase culture, both floating and adherent cells were analyzed by FACS.DiI⁺CD45⁺ hematopoietic cells (shown by “}”) were generated from theDiI-labeled endothelial-like cells. CD45⁺ cells in the adherent cellfraction are likely to be cobble stone-forming cells and cells attachedto the stromal cell layer.

FIG. 2A is a diagram representing cloning and expression of mouse PCLP1,showing FACS staining of LO cells with 10B9 monoclonal antibody (shadedpeak) or isotype control (blank peak).

FIG. 2B is a diagram representing cloning and expression of mouse PCLP1,showing FACS staining of COS7 cells transfected with mouse PCLP1 cDNA(shaded peak) or mock vector (blank peak) with 10B9 antibody.

FIG. 3 is a schematic representing an alignment of amino acid sequencesof mouse, human, and rabbit PCLP1, and avian thrombomucin. The underlineand double-underlines represent signal peptides and transmembranedomains, respectively. In human PCLP1, the reported signal peptidecontains two extra amino acid residues in addition to the underlinedresidues ( . . . LP).

FIG. 4 is a photographic representation the results of Northern blotanalysis of PCLP1 mRNA in various mouse adult tissues and LO cells.PolyA⁺ RNA (1 μg) was loaded in each lane.

FIG. 5A is a photographic representation showing the expression of PCLP1in an AGM culture: morphological appearance of endothelial-like cells inthe AGM primary culture on day 6. Original magnification was 100×.

FIG. 5B is a photographic representation of the immunostaining ofendothelial-like cells in the AGM culture with isotype control (B).Original magnification was 200×.

FIG. 5C is a photographic representation of the expression of PCLP1 inan AGM culture using 10B9 anti-PCLP1 antibody (C). Originalmagnification: 200×.

FIG. 6 is a diagram representing the expression of PCLP1 and CD45 in anAGM culture.

Represents a FACS analysis of the total cells in the AGM culture on day6 with anti-PCLP1 and anti-CD45 antibodies. R2 gated cells were sortedas a PCLP1⁺CD45⁻ fraction for the co-culture experiment shown in FIG. 7.

FIG. 7 is a diagram representing the result of the FACS analysis of thefloating cells and adherent cells in a co-culture (middle panel) wherethe PCLP1⁺CD45⁻ cells from the AGM culture of GFP mice were sorted onday 6 and co-cultured with the AGM culture of normal mice for 4 moredays. Upper and bottom panels are negative (AGM culture of normal mice)and positive (AGM culture of GFP mice) controls for the detection ofGFP, respectively. Note that GFP⁺CD45⁺ cells are generated fromPCLP1⁺CD45⁻ cells in the co-culture.

FIG. 8A is a diagram representing the result of FACS analyses of the AGMregion of mouse embryos, showing the expression of PCLP1 and CD45 incells from the AGM region. A single cell suspension prepared from theAGM regions of mouse embryos at 11.5 dpc was stained with anti-CD45antibody and anti-PCLP1 antibody or isotype control and analyzed byFACS.

FIG. 8B is a diagram representing the result of FACS analyses of the AGMregion of mouse embryos, showing the expression of CD31 and CD34 in theCD45⁻ cell population. The AGM-derived cells were stained withanti-PCLP1 antibody and anti-CD45 antibody together with eitheranti-CD34 or anti-CD31 antibodies. FACS profiles of PCLP1 and CD34 orCD31 in the CD45⁻ cell fraction are shown. Note that most of CD34⁺ andCD30⁻ cells are included in the PCLP1⁺CD45⁻ cell fraction.

FIG. 9A is a diagram representing the result of FACS analysis of the AGMregion of mouse embryos, showing the expression of Flk1 and VECadherinin the CD45⁻ cell population. The AGM-derived cells were stained withanti-PCLP1 and anti-CD45 antibodies together with anti-Flk1 antibody.Expression patterns of PCLP1 and Flk1 in the CD45⁻ cell fraction arepresented.

FIG. 9B is a diagram representing the result of FACS analysis of the AGMregion of mouse embryos, showing the expression of Flk1 and VECadherinin the CD45⁻ cell population. The AGM-derived cells were stained withanti-PCLP1 and anti-CD45 antibodies together with anti-VECadherinantibody. Expression patterns of VECadherin in the CD45⁻ cell fractionare presented.

FIG. 10 is a photographic representation showing an expression of PCLP1in the AGM region of a mouse embryo. Paraffin sections of the AGM regionof a mouse embryo at 11.5 dpc were stained immunohistochemically withisotype control (A to C), anti-PCLP1 (D to F), or anti-CD34 (G to I)antibodies. Expression of PCLP1 and CD34, shown in brown, mostly overlapin the aorta (E, H) and genital ridge regions (F, I).

FIG. 11 is a photographic representation showing an expression of PCLP1in the AGM region of a mouse embryo. Paraffin sections of the AGM regionwere subjected to in situ hybridization using sense (J to L) oranti-sense (M to O) cRNA to the mouse PCLP1 cDNA as a probe. Specificsignals are shown in dark blue. Original magnification was 40× (leftpanels). Aorta and genital ridge regions are further enlarged in middleand right panels, respectively.

FIG. 12 is a diagram representing differentiation of PCLP1⁺CD45⁻ cellsinto endothelial cells.

Shows the FACS analysis of the cells in the AGM regions of mouse embryosat 11.5 dpc with anti-PCLP1 and anti-CD45 antibodies. In lower panels,sorted PCLP1⁺CD45⁻ cells (R1 gate) or PCLP1⁻-CD45⁻ cells (R2 gate) werereanalyzed, respectively.

FIG. 13 is a photographic representation differentiation of PCLP1⁺CD45⁻cells into endothelial cells. Morphological appearance of the sortedPCLP1⁺CD45⁻ cells from the AGM region after 6 days in culture with SCFand OSM.

FIG. 14A is a diagram representing differentiation of PCLP1⁺CD45⁻ cellsinto endothelial cells, showing the incorporation of DiI-labeledacetylated LDL into PCLP1⁺CD45⁻ cells after 6 days in culture. Shadedand blank peaks represent FACS patterns of cells incubated with orwithout DiI-acetylated LDL, respectively.

FIG. 14B is a diagram representing differentiation of PCLP1⁺CD45⁻ cellsinto endothelial cells, showing the expression of Flk1 on thePCLP1⁺CD45⁻ cells after 6 days in culture. Cells were stained withanti-Flk1 (shaded peak) or isotype control (blank peak) antibody andsubjected to FACS analysis.

FIG. 15 is a diagram representing differentiation of PCLP1⁺CD45⁻ cellsinto endothelial cells. Induction of the expression of variousendothelial cell markers in PCLP1⁺CD45⁻ cells after co-culture with OP9stromal cells for 10 days in the presence of OSM, VEGF, and bFGF. OP9cells were gated out by the forward scatter window and the remainingmajor cell fraction was stained with antibodies as indicated. Leftpanels demonstrate FACS patterns of the PCLP1⁺CD45⁻ cells after 6 daysin culture in the absence of OP9. Blank and light shaded peaks showstaining patterns of isotype control and specific antibodies,respectively.

FIG. 16 is a photographic representation showing differentiation ofPCLP1⁺CD45⁻ cells into endothelial cells.

Shows vascular network formation of the endothelial cells. After 10 daysin co-culture of the PCLP1⁺CD45⁻ cells with OP9, cells were placed onthe matrigel and cultured for 12 hours.

FIG. 17 is a diagram representing generation of hematopoietic cells fromPCLP1⁺CD45⁻ cells in vitro. The PCLP1⁺CD45⁻ cells isolated from the AGMregion were co-cultured with OP9 stromal cells in the presence of SCF,bFGF, OSM, LIF, IL-3, and EPO for 10 days. Floating cells weredouble-stained with two anti-hematopoietic lineage marker antibodies orisotype controls as indicated and subjected to FACS analysis.

FIG. 18 is a diagram representing induction of long-term repopulatinghematopoietic stem cells in vivo from PCLP1⁺CD45⁻ hemangioblasts.GFP⁺PCLP1⁺CD45⁻ cells were isolated from the AGM region of the GFPtransgenic mice and 1.7×10⁵ cells were injected into the liver ofneonatal C57BL/6 mice at 36 h after birth. After 6 months, peripheralblood was taken from one mouse (10B3 mouse) and mononuclear cells werestained with various antibodies against hematopoietic lineage markers asindicated. FACS patterns of C57BL/6 and GFP mouse are shown as negativeand positive controls, respectively. Note the higher contribution of thedonor-derived cells in the hematopoietic system of the 10B3 mouse.

FIG. 19 is a diagram representing the induction of long-termrepopulating hematopoietic stem cells from PLCP1⁺CD45⁻ hemangioblasts invivo. Spleen was extracted from a similarly transplanted mouse as inFIG. 18, and mononuclear cells were stained with various antibodiesagainst hematopoietic lineage markers as indicated.

FIG. 20 is a diagram representing the induction of long-termrepopulating hematopoietic stem cells from PLCP1⁺CD45⁻ hemangioblasts invivo. Bone marrow was collected from a similarly transplanted mouse asin FIG. 18, and mononuclear cells were stained with various antibodiesagainst hematopoietic lineage markers as indicated.

FIG. 21 is a diagram representing the induction of long-termrepopulating hematopoietic stem cells from PLCP1⁺CD45⁻ hemangioblasts invivo. Thymus was extracted from a similarly transplanted mouse as inFIG. 18, and mononuclear cells were stained with various antibodiesagainst hematopoietic lineage markers as indicated. Analysis of thymusfrom 10B3 mouse was first performed gating GFP⁺ thymocytes to analyzethe expression of CD4 and CD8.

DESCRIPTION

The present invention will be explained in detail below with referenceto Examples, but it is not to be construed as being limited thereto.

Timed pregnant C57BL/6 mice were purchased from Nihon SLC (Hamamatsu,Japan). GFP transgenic mice (Okabe, M. et al. (1997) FEBS Lett. 407,313-319) were maintained and mated in an animal facility. The time atmidday (12:00) was taken to be 0.5 dpc for the plugged mice. Aspreviously described (Mukouyama, Y et al. (1998) Immunity 8, 105-114),AGM regions were dissected from mouse embryos at 11.5 dpc and a singlecell suspension was subjected to primary culture.

Flow cytometry and cell sorting conducted in Examples are describedbelow. Isolated AGM regions were dissociated by incubation with dispase(Boehringer) for 30 minutes at 37° C. and cell dissociation buffer(Gibco-BRL) for 30 minutes at 37° C., followed by vigorous agitation toseparate cells. Single cell suspensions of the AGM culture were preparedby incubating with cell dissociation buffer for 30 minutes at 37° C.

Cells were first incubated with 50 μl of mouse serum on ice for 30minutes and biotinylated primary antibody was added at 10 μg/ml. After a30 minute incubation on ice, a 20-fold volume of phosphate bufferedsaline at pH 7.4 (PBS) containing FCS was added and the cells werecentrifuged. Cells were then incubated with allophycocyanin(APC)-conjugated streptavidin (Molecular probe, Eugene, Oreg.) at 10μg/ml for 30 minutes on ice with or without phycoerythrin(PE)-conjugated antibody. After washing with 5% FCS-PBS, cells wereresuspended in 0.5 ml of PBS containing propidium iodide (PI) (Sigma,St. Louis, Mo.) and analyzed by FACS Calibur (Becton Dickinson).PI-positive dead cells were excluded. The monoclonal antibodies used forFACS were anti-CD45 (30F11.1), anti-Mac-1 (M1/70), anti-Gr-1 (RB6-8C5),anti-Thy-1.2 (30-H12), anti-B220 (RA3-6B2), anti-Ter-119 (TER-119),anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-CD34 (RAM34), anti-CD31(MEC13.3), anti-Flk1 (Avas12□1), and rat isotype control (R35-95), whichwere all purchased from Pharmingen. Anti-VECadherin antibody (VECD1)(Matsuyoshi, N. et al. (1997) Proc. Assoc. Am. Physicians 109, 362-371)was kindly provided by S. Nishikawa (Kyoto University).

For cell sorting, AGM regions from GFP positive embryos at 11.5 dpc weretrypsinized as described above and cells (107/ml) were incubated withbiotinylated anti-PCLP1 antibody at 10 μg/ml in 5% FCS-PBS on ice for 30minutes. After washing with 20-fold volumes of 5% FCS-PBS, cells werestained with PE-conjugated anti-CD45 antibody (10 μg/ml) andAPC-conjugated streptavidin (10 μg/ml) on ice for 30 minutes andsubjected to cell sorting using FACS Vantage. In a typical case of cellsorting in combination with anti-PCLP1 and anti-CD45 antibodies asdescribed below, out of 1.1×107 cells obtained from 40 AGM regions,8.5×105 of PCLP1+CD45− cells, 1.0×106 of PCLP1−CD45− cells, and 5.9×104of PCLP1+CD45+ cells were obtained by cell sorting.

EXAMPLE 1 Generation of Hematopoietic Cells in the AGM Culture

The inventors' previous studies using an in vitro culture for AGM cells,they suggested that the endothelial-like cells in a AGM culture maycontain hemangioblasts which give rise to hematopoietic progenitors invitro (Mukouyama, Y. et al. (1998) Immunity 8, 105-114). Furthermore,timelap analysis of the cultured AGM cells under a phase contrastmicroscope showed that floating round cells with a hematopoieticappearance were spontaneously generated from adherent endothelial-likecells in situ (data not shown). To test the possibility that theadherent endothelial-like cells produced hematopoietic cells, theinventors examined the uptake of DiI-Ac-LDL, which is known to beincorporated only into endothelial cells and macrophages (Goldstein, J.L. et al. (1979) Proc. Natl. Acad. Sci. USA 76, 333-337; Voyta, J. C. etal. (1984) J. Cell Biol. 99, 2034-2040). As shown in FIG. 1, theinventors first incubated AGM cells for 6 days to generateendothelial-like cells. AGM cells at day 6 were washed well with theculture medium to remove hematopoietic cells, and co-incubated with 10μg/ml of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindo-carbocyanineperchlorate (Biomedical Technologies, Inc., Stoughton, Mass.)-labeledacetylated low density lipoprotein (DiI-Ac-LDL) at 37° C. for 6 hours.After washing twice with PBS, AGM cells were stained with anti-CD45antibodies (Pharmingen, San Diego, Calif.) conjugated fluoresceinisothiocyanate (FITC). DiI⁺CD45⁻ cell population was sorted by FACSVantage (Becton Dickinson, Bedford, Mass.) and inoculated to unlabeledAGM culture at day 6. DiI⁺CD45⁺ hematopoietic cells appeared after 4days of co-incubation (FIG. 1). These DiI⁺CD45⁺ hematopoietic cells weresorted and subjected to CFU-C assay.

CFU-C assay was conducted as follows: Cells (10⁸³⁶ ⁴) were inoculatedinto 0.8% methylcellulose medium containing 20% fetal calf serum, IL-3(100 ng/ml), IL-6 (kind gift from Ajinomoto, Kawasaki) (100 ng/ml), SCF(kind gift from Kirin Brewery, Takasaki, Japan) (100 ng/ml), and EPO(kind gift from Kirin Brewery) (2 U/ml) and cultured for 14 days aspreviously described (Mukouyama, Y et al. (1998) Immunity 8, 105-114).These hematopoietic cells formed colonies in the CFU-C assay (data notshown), suggesting that some hematopoietic progenitor cells were derivedfrom the DiI⁺CD45⁻ endothelial-like cells in the AGM primary culture.

EXAMPLE 2 Preparation of Monoclonal Antibodies Against Surface Antigensof Endothelial-Like Cells Derived from AGM Culture

To define hemangioblasts more precisely, the inventors aimed to obtain aspecific antibody directed against hemangioblasts. By repeating thepassage of adherent cells of the AGM culture in the presence of OSM, theinventors were able to establish a novel OSM-dependent endothelial-likecell line, LO. LO cells exhibit characteristics very similar to those ofendothelial-like cells in the AGM culture, such as endothelial-likemorphology, incorporation of DiI-Ac-LDL, and production of hematopoieticcells. The inventors used the LO cells as immunogens to raise monoclonalantibodies against cell surface antigens on LO cells as follows.

Wistar rats (Nihon SLC) were immunized with 10⁷ of LO cells in thepresence of Freund's complete adjuvant (WAKO, Osaka, Japan) according tothe standard immunization procedure (Hockfield, S. et al. (1993)“Selected Methods for Antibody and Nucleic Acid Probes”, Volume 1 (NewYork: Cold Spring Harbor Laboratory Press)). Lymph nodes weredissociated and fused with mouse myeloma P3X cells using polyethyleneglycol as previously described (Ogorochi, T. et al. (1992) Blood 79,895-903) and hybridoma supernatants were screened for the production ofanti-LO specific antibodies by FACS. 10B9 monoclonal antibody was chosenbased on the specific staining of endothelial-like cells in the AGMculture. 10B9 antibody was produced in nude mice and purified by usingE-Z-Sep (Pharmacia Biotech, Uppsala, Sweden). The isotype of the 10B9antibody was determined by using the rat IgG isotyping kit (Serotec,Oxford, UK). Biotinylated 10B9 antibody was prepared by using Enzotin(Enzo Diagnostics, Syosset, N.Y.) according to the manufacturer'sinstruction.

Flow cytometry revealed that the antibody designated 10B9 (rat IgG1)exhibited very clear staining of LO cells (FIG. 2A) but not of NIH3T3cells (data not shown). This antibody also stained endothelial-likecells in the AGM culture as described below (see FIG. 5).

EXAMPLE 3 Molecular Cloning of Mouse PCLP1 Molecule as a PossibleHemangioblast Antigen

Next, using a standard expression cloning strategy with COS7 cells and10B9 monoclonal antibody, the inventors isolated a cDNA clone encodingthe 10B9 antigen.

Expression cloning of a cDNA encoding the 10B9 antigen was carried outby using COS7 cells as previously described (Harada, N. et al. (1990)Proc. Natl. Acad. Sci. USA 87, 857-861) except that magnetic beadsconjugated with anti-rat IgG antibody (Dynabeads M-450) (Dynal, Oslo,Norway) were employed instead of plate panning. Briefly, COS7 cells werefused with spheroplasts of the cDNA plasmid library of LO cells (Tanaka,M. et al. (1999) Blood 93, 804-815) and stained with 10B9 antibodyfollowed by Dynabead selection. Plasmid DNA mixture was harvested fromthe beads, amplified in E. coli and re-transfected into COS7 cells. Thisprocedure was repeated 4 to 5 times until a single band of cDNA insertwas recovered. As a result, the inventors isolated a cDNA clone of 1.9kilobases encoding the 10B9 antigen.

DNA sequencing revealed that the C-terminal amino acid sequence washighly homologous to those of human and rabbit podocalyxin-like protein1 (PCLP1) (Kershaw, D. B. et al. (1997) J. Biol. Chem. 272, 15708-15714;Kershaw, D. B. et al. (1995) J. Biol. Chem. 270, 29439-29446),suggesting that it was a mouse counterpart of PCLP1 (FIG. 3). The avianPCLP1 homolog, thrombomucin, also shares the conserved regions (McNagny,K. M. et al. (1997) J. Cell Biol. 138, 1395-1407) (FIG. 3). To obtainthe full-length mouse PCLP1 cDNA, the inventors isolated 5′ cDNAfragments of mouse PCLP1 through screening of the original cDNA libraryand rapid amplification of the cDNA ends (RACE) method. 5′-RACE wasperformed using the 5′-RACE kit (GIBCO-BRL). The DNA sequences of thecDNAs were determined by using a Dye terminator cycle sequencing kit(Perkin Elmer, Foster City, Calif.) and an automated DNA sequencer(Applied Biosystems, Foster City, Calif.). The cDNA nucleotide sequenceof mouse PCLP1 and the amino acid sequence of protein encoded by thecDNA are set forth in SEQ ID NO: 1 and 2, respectively.

COS7 cells were transfected with the reconstructed full-length mousePCLP1 cDNA in the pME18S expression vector and were stained with 10B9antibody. The COS7 cells transfected with PCLP1 cDNA exhibited specificstaining with 10B9 antibody (FIG. 2B), confirming that the 10B9 antibodyrecognizes mouse PCLP1. PCLP1 is an extensively glycosylated proteinwith a single transmembrane region. As previously reported (Kershaw, D.B. et al. (1997) J. Biol. Chem. 272, 15708-15714; Kershaw, D. B. et al.(1995) J. Biol. Chem. 270, 29439-29446), the amino acid sequence of theN-terminal region of PCLP1 is poorly conserved among species (FIG. 3).Interestingly, a recent report suggested that both PCLP1 and CD34 areligands for L-selectin in the high endothelial venule and that PCLP1 andCD34 share common amino acid sequences in their cytoplasmic tails(Sassetti, C. et al. (1998) J. Exp. Med. 187, 1965-1975). Thesehomologous amino acid residues are also found in mouse PCLP1 atpositions 440 to 451, 464 to 473 and 500 to 503 (FIG. 3).

For Northern blotting, poly(A)+ RNA samples were electrophoreticallyseparated in 1.0% agarose gel and transferred onto a nylon membrane(Boehringer Mannheim, Mannheim, Germany). The RNA was then hybridizedwith digoxigenin (DIG)-labeled single strand DNA probe for the PCLP1cDNA (2.1 kb) as described previously (Tanaka, M. et al. (1999) Blood93, 804-815)

PCLP1 was originally identified as a major component of podocytes in therabbit kidney and demonstrated to be expressed in some endothelial cells(Kershaw, D. B. et al. (1995) J. Biol. Chem. 270, 29439-29446).Consistent with previous reports (Kershaw, D. B. et al. (1997) J. Biol.Chem. 272, 15708-15714; Kershaw, D. B. et al. (1995) J. Biol. Chem. 270,29439-29446), the inventors detected PCLP1 mRNA in kidney, heart, lung,brain, and muscle, but not in spleen, thymus, small intestine, or liverof adult mice (FIG. 4). The same size of mRNA was also detected in LOcells (FIG. 4). The avian counterpart of PCLP-1, thrombomucin, wasreported to be expressed in thrombocytes and multipotent hematopoieticprogenitors (McNagny, K. M. et al. (1997) J. Cell Biol. 138, 1395-1407).Likewise, expression of PCLP1 was found in some bone marrow cells (datanot shown) and hematopoietic cells in the AGM region (see FIG. 6, 8A) asdescribed below.

EXAMPLE 4 Expression of PCLP1 on the Endothelial-Like Cells in the AGMCulture

The inventors examined the expression of PCLP1 on the endothelial-likecells in the AGM culture by immunostaining with 10B9 anti-PCLP1antibody. Cultured AGM-derived cells in plastic plates were fixed with1% paraformaldehyde (PFA)-PBS at room temperature for 15 minutes andincubated with anti-PCLP1 10B9 antibody at 10 μg/ml at 4° C. over night.After incubation with peroxidase-conjugated anti-rat IgG (Amersham),signals were visualized by 3,3′-diaminobenzidine (DAB) as previouslydescribed (Hara, T. et al. (1998) Dev. Biol. 201, 144-153).

As the inventors expected, PCLP1 was detectable on endothelial-likecells (FIG. 5A to C), but not on fibroblastic cells (data not shown) inthe AGM culture. The endothelial-like cells, defined by their polygonalcell morphology and incorporation of DiI-Ac-LDL, were furtherfractionated by fluorescent activated cell sorting (FACS) usinganti-PCLP1 and anti-CD45 antibodies (FIG. 6). Except for the erythroidlineage, CD45 is known to be a pan specific marker for hematopoieticcells including LTR-HSCs (Morrison, S. J. et al. (1995) Annu. Rev. Cell.Dev. Biol. 11, 35-71). It is noteworthy that hematopoietic cells (CD45⁺)in the AGM culture also express a high level of PCLP1 (FIG. 6) as is thecase for the AGM region (FIG. 8A).

To examine whether the PCLP1⁺CD45⁻ non-hematopoietic fraction containshemangioblasts, PCLP1⁺CD45⁻ cells (2×10⁵) were isolated (the R2 gateshown in FIG. 6) from the day 6 AGM culture of transgenic miceexpressing green fluorescent protein (GFP) and were inoculated into theday 6 AGM culture of nontransgenic mice. After 4 days of incubation,GFP⁺CD45⁺ hematopoietic cells appeared in both floating and adherentfractions (FIG. 7), indicating that hematopoietic cells are generatedfrom the PCLP1⁺CD45⁻ endothelial-like cells in the AGM primary culture.The adherent GFP⁺CD45⁺ cells may represent the hematopoietic cellspresent underneath the stromal cell layer.

EXAMPLE 5 Expression and localization of PCLP1 in the AGM Region of aMouse Embryo

The inventors next examined the presence of the PCLP1⁺CD45⁻ cells in theintact AGM region of mouse embryos at 11.5 dpc. Based on FACS staining,there were more PCLP1⁺CD45⁻ cells (32%) than PCLP1⁺CD45⁺ cells (1.5%) inthe AGM region (FIG. 8A). The sorted PCLP1⁺CD45⁻ cells were adherentcells with the capacity to incorporate DiI-Ac-LDL (data not shown),indicating that these cells are endothelial-like cells. It was recentlyreported that hematogenic angioblasts in the yolk sac and the P-Spregion express Flk1, VECadherin, and CD34 (Nishikawa, S. I. et al.(1998) Immunity 8, 761-769). Thus, the inventors examined whether thesemolecules are expressed in the PCLP1⁺CD45⁻ fraction. Interestingly, amajority of nonhematopoietic CD34⁺ cells, CD31⁺ cells, and Flk1⁺ cellsalso expressed PCLP1 (FIG. 8B, 9A), whereas 79% of VECadherin⁺CD45⁻cells were found in the PCLP1⁻ fraction (FIG. 9B). Consistent with theoverlapping expression patterns of PCLP1 and CD34, expression of thesetwo proteins was localized in the endothelium of the dorsal aorta (FIG.10E, 10H) and in the genital ridge region (FIG. 10F, 10I) of the mouseembryo at 11.5 dpc.

In situ hybridization analysis of the paraffin sections of a mouseembryo was conducted as follows. For preparation of paraffin sections,the caudal half of mouse embryos at 11.5 dpc was fixed in 4% PFA-PBS for10 hours. Paraffin sections (6 μm thick) were prepared as previouslydescribed (Hara, T. et al. (1998) Dev. Biol. 201, 144-153) and placed onpoly-L-lysine-coated slide glasses. After hydration of paraffinsections, the sections were stained with anti-PCLP1 or anti-CD34antibody at 10 μg/ml at 4° C. over night and visualized as describedabove. Samples were counterstained with methylgreen.

In situ hybridization of the parrafin sections was carried out aspreviously described (Imakawa, K. et al. (1995) Endocrine 3, 511-517).DIG-labeled antisense and sense RNA probes were prepared by using the5′-part of the PCLP1 cDNA fragment (nucleotide 126 to 354).

In situ hybridization analysis of the PCLP1 mRNA in the paraffinsections of a mouse embryo also revealed a similar expression pattern inthe dorsal aorta (FIG. 11N) and the genital ridge (FIG. 11O). Sinceendothelial-like cells in the day 6 AGM culture do not expressVECadherin and CD34 (data not shown), the inventors employed PCLP1 as amarker for the separation of hemangioblasts in the AGM region in thefollowing Examples.

EXAMPLE 6 Endothelial Differentiation of the PCLP1⁺CD45⁻ Cells from theAGM Region

The PCLP1⁺CD45⁻ cell fraction was separated by cell sorting from the AGMregion of mouse embryos at 11.5 dpc (the R1 gate in FIG. 12). The sortedcells were reanalyzed, but CD45⁺ cells were undetectable (FIG. 12). Even3 hours after plating of the sorted PCLP1⁺CD45⁻ cells, nohematopoietic-like cells could be detected by microscopic observationand the cells were capable of incorporating DiI-Ac-LDL (data not shown).When these cells were cultured in the presence of OSM for 6 days,endothelial-like cells increased by 10-folds during incubation (FIG.13), incorporated DiI-Ac-LDL and expressed Flk1 (FIG. 14 top andbottom), whereas no cells grew in the absence of OSM. Since only 12% ofthe sorted PCLP1⁺CD45⁻ cells were Flk1⁺ cells at the time of separation(FIG. 9A), Flk1⁺ cells may be selectively expanded or Flk1 expressionmay be induced during cultivation. The PCLP1⁺CD45⁻ cells grown in thepresence of OSM were partially positive for CD31, negative for CD34, andvery weakly positive for VECadherin (FIG. 15).

To test the possibility that the PCLP1+CD45− cells differentiate toendothelial cells, the inventors employed the OP9 co-culture system thathas been used to induce endothelial differentiation in vitro (Hamaguchi,I. et al. (1999) Blood 93, 1549-1556; Hirashima, M. et al. (1999) Blood93, 1253-1263).

Mouse calvaria-derived OP9 cells (kindly provided by S, Nishikawa, KyotoUniversity) were passaged as previously described (Kodama, H. et al.(1994) Exp. Hematol. 22, 979-984). Sorted PCLP1⁺CD45⁻ cells from the AGMregion were inoculated on subconfluent OP9 cells in a AGM culture mediumcontaining various cytokines and cultured for 10 days. For thegeneration of hematopoietic cells, 5×10⁴ cells were co-cultured with OP9in the presence of SCF (100 ng/ml), bFGF (1 ng/ml), LIF (10 ng/ml), OSM(10 ng/ml), IL-3 (10 ng/ml), and EPO (2 U/ml). For endothelial celldifferentiation, 10⁴ cells were co-cultured in the presence of OSM (10ng/ml), bFGF (1 ng/ml), and VEGF (PeproTech, London, UK) (10 ng/ml).

For matrigel assays, cells (2×105) were resuspended in Dulbecco'smodified Eagle's medium containing 1% fetal calf serum and VEGF (10ng/ml) and overlayed on a Biocoat matrigel basement membrane (BectonDickinson) in a 6-well plate. After 12 hours in culture, networkformation was microscopically observed.

The PCLP1⁺CD45⁻ cells from the AGM region were co-cultured with OP9stromal cells for 10 days in the presence of OSM, VEGF, and bFGF asdescribed above. The resultant cells expressed higher levels of CD34 andVECadherin (FIG. 15) than those in the initial cell population (FIG. 8B,9B) or those cultured with OSM alone. Co-culture of the PCLP1⁺CD45⁻cells with OP9 also resulted in an increased expression of CD31 and adecreased expression of PCLP1 (FIG. 15). Moreover, the OP9 co-culturedcells formed a vascular network on a matrigel plate (FIG. 16), while thecells grown without OP9 failed to form a network (data not shown). Theseresults indicate that PCLP1⁺CD45⁻ cells in the AGM region are able todifferentiate to endothelial cells in the presence of OP9, OSM, VEGF,and bFGF. Therefore, PCLP1⁺CD45⁻ cells are likely to be the endothelialprecursor cells, i.e. angioblasts. Growth of the angioblasts in the AGMregion appears to be OSM-dependent and their differentiation requiresadditional factors including VEGF, bFGF, and unknown factors producedfrom OP9 cells.

On the other hand, co-culture of PCLP1⁺CD45⁻ cells with OP9 cells in thepresence of hematopoietic growth factors containing SCF, interleukin(IL)-3, and erythropoietin (EPO) resulted in the development ofhematopoietic cells. The hematopoietic cells included Mac-1/Gr-1positive myeloid cells, B220/Thy-1-positive lymphoid cells, and Ter19-positive erythroid cells (FIG. 17), suggesting that multiple lineagesof hematopoietic cells were generated from the PCLP1⁺CD45⁻ cells invitro. Generation of these hematopoietic cells was also OSM-dependent.Taken together with the data from the DiI-Ac-LDL labeling experiment(FIG. 1), it can be concluded that PCLP1⁺CD45⁻ cells in the AGM regioncontain hemangioblasts and angioblasts.

EXAMPLE 7 Generation of LTR-HSCs from the PCLP1⁺CD45⁻ Cells in the AGMRegion

A major goal of this Example was to know whether hemangioblasts in theAGM region could give rise to LTR-HSCs in vivo. LTR-HSCs were detectedamong the hematopoietic progenitors expanded in the AGM culture by thestandard repopulation assay using irradiated adult mice. However, it wasrevealed that they are more efficiently engrafted when injected intolivers of busulfan-treated neonatal mice (data not shown). This isreasonable as LTR-HSCs generated in the AGM region seed the fetal liverin vivo before homing into the bone marrow. The inventors thusconsidered the possibility that if LTR-HSCs were generated from thehemangioblasts present in the AGM region, they would engraft theneonatal liver more efficiently than in irradiated adult mice, andverified that. According to a recently established procedure (Yoder, M.C. et al. (1996) Biol. Blood Marrow Transplant. 2, 59-67), the inventorsinjected the PCLP1+CD45⁻ cells from the AGM regions of GFP transgenicmouse embryos at 11.5 dpc into the busulfan-treated nontransgenicneonatal mice.

Transplantation of cells into busulfan-treated neonatal mice wasperformed as previously described (Yoder, M. C. et al. (1997) Immunity7, 335-344) with a slight modification. Briefly, busulfan (Sigma) wasintraperitoneally injected into pregnant C57BL/6 mice at 12.5 □g/g onpregnant day 17 and 18. Within 24 to 48 hours after birth, cells derivedfrom GFP mice in 25 □l of PBS were injected into the liver of neonatalmice. Peripheral blood of recipient mice was taken at 2 or 6 monthsafter transplantation and analyzed for GFP chimerism.

As summarized in Table 1, donor-derived GFP positive hematopoietic cellswere detected in the peripheral blood of 7 out of 9 mice at 2 to 6months after the injection of the GFP+PCLP1+CD45− cells. To repopulatethe donor-derived blood cells, an injection of 1.7×105 cells or more wasrequired, indicating that a small fraction of the PCLP1+CD45− cellpopulation are capable of generating LTR-HSCs. In contrast, nodonor-derived hematopoietic cells were found by injecting the samenumber of PCLP1−CD45− cells from the AGM region (the R2 gate in FIG. 12)(Table 1). Moreover, injection of the PCLP1+CD45+ cells (0.9 to 2.1×104)did not contribute to GFP chimerism, which effectively excluded thepossibility that a small number of contaminating CD45+ cells in thePCLP1+CD45− fraction repopulated in the recipient mice. The chimerismwas maintained up to 6 months in both myeloid and lymphoid compartmentsof the peripheral blood of a mouse (10B3 mouse) injected with 1.7×105 ofthe PCLP1+CD45− cells (FIG. 18). In the 10B3 mouse, all lineages ofdonor-derived GFP positive hematopoietic cells were repopulated in thespleen (FIG. 19) and bone marrow (FIG. 20). CD4/CD8-double positive andmature single positive T cells derived from the donor were also detectedin the recipient thymus (FIG. 21). These results indicate thathemangioblasts with potential to generate LTR-HSCs are present in thePCLP1+CD45− cell population of the AGM region. Although a similar numberof the PCLP1+CD45− cells derived from the day 6 AGM primary cultureshowed a decreased repopulation potential (Table 1), they were capableof generating hematopoietic cells in vitro (FIG. 7). Hence, the in vitroculture of the AGM cells negatively affected the repopulation potentialof the PCLP1+CD45− cells.

TABLE 1 Hematopoietic Cell Generation from PCLP1+CD45− Cells inEngrafted mice Cell number/ Engrafted/Total Exp. Cell Fraction mouse (%Chimerism) 1 AGM PCLP1⁺CD45⁻ 1.7 × 10⁵ 1/1* (53%) PCLP1⁺CD45⁺ 9.0 × 10³0/1* 2 AGM PCLP1⁺CD45⁻ 2.8 × 10⁵ 2/2 (21%, 1.2%) PCLP1⁻CD45⁻ 2.8 × 10⁵0/3 3 AGM PCLP1⁺CD45⁻ 3.4 × 10⁵ 1/1 (29%) PCLP1⁻CD45⁻ 3.4 × 10⁵ 0/1 4AGM PCLP1⁺CD45⁻ 2.2 × 10⁵ 3/5 (4.4%, 2.6%, 1.5%) PCLP1⁺CD45⁺ 2.1 × 10⁴0/4 5 AGM PCLP1⁺CD45⁻ 3.0 × 10⁵ 2/5 (0.35%, 0.23%) culture PCLP1⁺CD45⁺2.9 × 10⁴ 0/2 6 AGM PCLP1⁺CD45⁻ 3.0 × 10⁵ 2/5 (0.14%, 0.14%) culturePCLP1⁻CD45⁻ 3.0 × 10⁵ 0/2

Each cell fraction was sorted from AGM region or AGM culture of GFP miceand injected into busulfan-treated neonatal mice. Peripheral blood wastaken at 2 months (6 months for those marked “*”) after injection andsubjected to FACS analysis. Relative frequency of GFP+ cells inengrafted mice was calculated and expressed as “% chimerism”.

The present invention provides a method for preparing a cell fractioncontaining hemangioblasts capable of generating both endothelial cellsand hematopoietic cells, and a marker molecule “PCLP1” forhemangioblasts utilized in the preparation. A cell fraction according tothe present invention is capable of not only differentiating intoendothelial-like cells and hematopoietic cells, but also of expressing along-term hematopoietic function in vivo. The method of this inventionenables the screening and separation of hemangioblasts in varioustissues and cells. A cell fraction of this invention is not only useful,for example, in screening factors and drugs that regulate theproliferation and differentiation of hematopoietic stem cells, but couldalso be used for isolating novel cell markers for hemangioblasts andhematopoietic stem cells, or for screening antibodies used in cellsorting.

1. A mouse-derived PCLP1 protein encoded by a DNA according to any oneof the following (a) through (c): (a) a DNA comprising the coding regionof the nucleotide sequence set forth in SEQ ID NO: 1, (b) a DNA encodinga protein comprising the amino acid sequence set forth in SEQ ID NO: 2,or (c) a DNA encoding a protein comprising the amino acid sequence setforth in SEQ ID NO: 2 in which one or more amino acids are substituted,deleted, inserted, and/or added.
 2. A polypeptide containing a partialsequence comprising at least seven or more consecutive amino acidresidues at positions 1 to 405 in the amino acid sequence set forth inSEQ ID NO: 2.